4.2 General Information

• 4.2.1 FDA Law and Filth
• 4.2.2 Microscopic Examination and Microscope Accessories
• 4.2.3 Preparing Microscopic Slide Mounts
• 4.2.4 Collecting and Preserving Whole Insects and Arthropods
• 4.2.5 Taxonomy
• 4.2.5.1 Taxonomy Principles
• 4.2.5.2 Identification Keys
• 4.2.6 Digital Photography and Photomicrography
• 4.2.7 Analytical Filth Worksheet

The trainees will receive Basic Orientation Training through ORA University web based modules, discussions, exercises, and videos;  the trainees are expected to complete the ORA University Analyst Bingo card within six months of employment.

Before beginning to work in a district laboratory, the trainee should be given a thorough safety orientation that includes familiarization with the laboratory's safety features and regulations, and the local laboratory's Standard Operating Procedures (SOP’s) and Quality Assurance Procedures.  Emphasis is placed towards the policies and procedures inherent in the filth analysis program.  Additional information can be found in the ORA Lab Manual, Volume III, Section 2, “Environmental Health and Safety.”

4.2.1 FDA Law and Filth

A. Objective

This exercise describes the legal basis for the agency’s regulatory activity in foods, drugs, and cosmetics contaminated (adulterated) with filth.
 
In order to analyze a filth sample, the analyst is to be intimately familiar with the requirements and prohibitions of the Food, Drug, and Cosmetic (FD&C) Act. The entomologist's analysis demonstrates compliance or non-compliance with sections of the Act. Entomologists work primarily with evidence collected to show non-compliance with Sections 402(a)(3), 402(a)(4), 501(a)(1), 501(a)(2), 601(b), and 601(c) of the FD&C Act. 
 
It is very important to have a clear understanding of the difference between sections 402(a)(3) and 402(a)(4). Section 402(a)(3) deals with a condition of the food that is objectionable in itself, whereas section 402(a)(4) deals with an objectionable practice. 
 
Sections 501(a)(1), 501(a)(2) deal with adulterated drugs and devices and 601(b), 601(c), deal with adulterated cosmetics for the same types of offenses as described above. 
 
A careful reading and discussion of the assigned literature is essential towards a more complete understanding of these and other sections of the FD&C Act.

B. Assignment

1. Complete:  Introduction to the FD & C Act, web based training module.
   
2. Read and discuss starred (*) items with the trainer from the following sections of the FD&C Act:  
   • 201(f)*, (g), and (i) Definitions
   • 301(a)*, (b)*, (c)*, (g), and (k)* Prohibited Acts
   • 402(a)(3)* and (a)(4)* Adulteration
   • 501(a)(1) and (a)(2)(A)
   • 601(b) and (c)
   • 702(b)* Reserve Sample
   • 704(d)* Letter
3. Read and discuss with the trainer:
   • Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, decomposition and foreign matter (FDA Technical Bulletin No. 1, 2nd ed., pp. 3-9).  Gaithersburg, MD: AOAC International.
   • Gentry, J. W., Harris, K., Gentry, Jr., J. W. (1991). Microanalytical entomology for food sanitation control (Vols. I and II, pp. 3-11). Gaithersburg, MD: AOAC International. 
   • Olsen, A. R., Sidebottom, T. H., Knight, S. A. (1996). Fundamentals of microanalytical entomology, a practical guide to detecting and identifying filth in foods (pp. 1-9). Boca Raton, FL: CRC Press. 
   • U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of Enforcement. Filth from Insects, Rodents, and Other Pests in Foods. Compliance policy guides, Sec. 555.600 (CPG 7120.18). http://www.fda.gov/ora/compliance_ref/cpg/cpgfod/cpg555-600.html. 
   • U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of Enforcement. Tree Nuts - Adulteration Involving Rejects (Insect Infestation, Moldy, Rancid, Otherwise Decomposed, Blanks, and Shriveled). Compliance policy guides, Sec. 570.425 (CPG 7112.05). http://www.fda.gov/ora/compliance_ref/cpg/cpgfod/cpg570-425.html.
   • U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of Enforcement. Foods, Adulteration Involving Hard or Sharp Foreign Objects. Compliance policy guides,Sec. 555.425. http://www.fda.gov/ora/compliance_ref/cpg/cpgfod/cpg555-425.htm.
   • U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of Enforcement.  Foods, Rail Car Sanitation – Adulteration. Compliance policy guides, Sec. 545.300 (CPG 7117.08).  http://www.fda.gov/ora/compliance_ref/cpg/cpgfod/cpg545-300.html.
   • U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition (May 1998 revision). The food defect action levels (read the Introduction). Retrieve version from  http://vm.cfsan.fda.gov/~dms/dalbook.html#intro.
   • U. S. Food & Drug Administration, Office of Regulatory Affairs, Office of Enforcement. Apricots - Canned - Adulteration with Insects. Compliance policy guides,Sec. 550.150 (CPG 7110.02). http://www.fda.gov/ora/compliance_ref/cpg/cpgfod/cpg550-150.html .

4.2.2 Microscopic Examination and Microscope Accessories

A. Objective

The trainee receives instruction towards the use and care of laboratory microscopes and their accessories, and learns proper terminology and definitions of the various forms of microscopic examination.  

B. Discussion

1. Types of Examinations and Microscopes
   
   1. Visual/Macroscopic Examination
      
      This type of product examination generally depends on the direct sensory input of the analyst. Examination is typically conducted with the naked eye but also may include the use of a hand lens (generally in the range of 3-10X), magnifying ring lamps (3X), or a pair of jeweler’s loupes (3-10X). 
   2. Widefield Stereomicroscope or Dissecting Microscope
       
      Results from a macroscopic examination may not be conclusive because the adulterant/defect can not be completely identified without the assistance of a stereo microscope.  This instrument, the most frequently used in filth analysis, has a wide variety of applications. The main application is for examining gross filth and for reading (examining) extraction papers for macroscopic filth. The most commonly used magnifications are from 6-30X; higher magnifications (50-75X) are used to confirm the identity of small objects. The microscopes exhibit a large field of view and have large working distances (from the lens to the focal point) in order to move and manipulate objects under the lens  
      
   3. Microscopic Examination 
       
      Filth/adulterants in the product are usually not visible to the naked eye and therefore are examined microscopically. A widefield stereo is often used first to manipulate and separate the contaminated product and then a compound microscope is used to positively identify the material and to see fine microscopic detail, morphology and sculpture. Compound microscopes optimize lighting techniques, and are engineered to manipulate light to see details not observable in stereoscopic examination, thus they have a very limited field of view and they have short working distances. The typical working magnifications are 100-400X, but may be used at 1000X with proper technique and special oil that helps capture and retain the light.
2. Fundamental Microscopic Techniques and Procedures
   
   1. Lighting and Ocular (eyepiece) Optimization Techniques
      
      Whether using a widefield or compound microscope, eye fatigue is usually the most important limiting factor when working with microscopes for any period of time. To help reduce headaches and eyestrain, the analyst needs to learn the following:
       
      Lighting:  Adjustment begins with the light source, which should be daylight, blue, or white (not frosted). Light strength should be adjusted with the transformer or iris diaphragm, so that details can be seen clearly with a minimum of glare and a minimum of intensity or brightness. For a stereomicroscope, the light source should be positioned slightly to one side and at approximately a 70° angle from horizontal, or overhead, as with fiberoptic ringlights. The light field should be centered so that both eyes receive the same intensity of light (i.e. balanced).
      
      Oculars (eyepieces) are adjusted to the individual analyst's eyes and facial features- while the eyes are in their most relaxed state.  This procedure should be done before each magnification.  Note: Leave both eyes open while adjusting the oculars.  Wearing eyeglasses is a matter of individual preference. People with astigmatism may find wearing their eyeglasses will reduce eye fatigue. If eyeglasses are to be worn, high eyepoint eyepieces may be ordered and should be used, and the ocular adjustments should be made with the eyeglasses on.
      
      Procedure:
      • Adjust the interpupillary distance of the oculars so that the oculars are centered on the respective pupils. 
      • Determine which ocular is independently focusable.
      • Place a specimen on the stage of the instrument, focus to the clearest image, and center it in the field of view. 
      • Hold a black or white index card between the focusable ocular and the eye, or simply remove the eyepiece, blocking vision on that side.  Do not close the eye or squint; the eye remains in a relaxed eye position.
      • Using the main focusing adjustment knob, clearly focus the image of the specimen for the eye that is not blocked. 
      • Remove the index card from the first position and use it to block the vision of the other eye (or remove the opposite eyepiece.) Using the focusing ring mechanism of the focusable ocular (not the main adjustment knob), clearly focus the image of the specimen.
      • Remove the card and using both eyes, view the specimen. The image should now be clearly in focus for both eyes in their most relaxed state
   2. Resolution (compound microscopes)
      
      For compound microscopes, the trainee should learn how to achieve optimal or Köehler illumination. The Köehler Principle focuses the field iris in the same plane as the specimen, thereby obtaining maximum resolution. Step-by-step instructions for achieving Köehler illumination are found in "Training Manual for Analytical Entomology in the Food Industry," Chapter 2 Part III D; FDA Technical Bulletin No. 2 and/or in the microscope manuals. The trainer demonstrates how to accomplish Koehler illumination and the trainee repeats the work on their unit.
   3. General Microscope Maintenance
       
      The analyst is responsible for simple maintenance procedures when using the instrument e.g. tightening loose focusing mechanisms, properly cleaning the lenses, and changing the light bulbs.  These and other maintenance operations are described in the instruction manuals accompanying each instrument, and should be addressed in the laboratory’s Standard Operating Procedures.  Maintenance records should be kept in the instrument logbook, and more complicated maintenance is directed to the assigned microscope monitor’s attention, and should not be attempted without prior approval.
3. Special Types of Microscopy and Accessories
   1. Phase-Contrast Microscope
      
      The phase-contrast microscope is a compound microscope that has special sub-stage accessories and objective lenses designed to produce optical contrast between the specimen and the mounting medium. Using phase-contrast optics, the analyst can observe many details that are obscure or indiscernible under a conventional compound microscope. This type of microscope is widely used for examining mites and small, somewhat transparent insects, like maggots, and in glass identification.
       
      Success with the phase-contrast microscope begins with and is highly dependent on the refractive indices of the specimen and the mounting medium. In order to get good contrast, the mounting medium should have the greatest possible difference in refractive index from the specimen. The difference between the refractive indices creates variations in light intensity. To the observer, it appears that a halo of intense light is surrounding the very dark edges and surface structures of the specimen, starkly silhouetting otherwise vague details.
       
      The instrument is adjusted in the same manner as a conventional compound microscope. Once optimal illumination is achieved, the phase-contrast optics are aligned in the following manner:
      
      1. Each phase objective lens has a metal ring of a defined size imbedded between two of the lens elements. The ring size is indicated by a number (usually "Ph 1," "Ph 2," or "Ph 3") printed on the lens casing.
      2. Similarly, the condenser has annular rings glued to a rotating clear plate that rotates into set position They are marked "1," "2," "3," and "clear (or J)" This latter position is the “normal” position for transmitted light microscopy, or the “starting" position for phase microscopy.
      3. In phase microscopy, the analyst properly selects the matching objective and condenser ring numbers, positioning the ring of the condenser in the light path, so that it will just encircle the ring in the objective. That is, the analyst pairs the objective ring to the condenser ring.  
          
      Initially, the adjustment of the instrument proceeds in the same manner as conventional transmitted light microscopy, using the “J” condenser position. Establish Köehler illumination first, and then focus on a mounted slide object (like a mite’s hairy leg). Finally, select the low power objective and begin aligning the phase-contrast optics, i.e. each objective with its corresponding condenser ring. Phase-contrast microscopes vary significantly by manufacturer and the proper alignment techniques are unique to each (some use focusing eyepieces, while others use swing-in focusing lenses). Therefore, consult the manual for the particular microscope, or have the trainer demonstrate how to critically align the rings.
   2. Polarized Light Microscopy (PLM)
       
      A polarizing light microscope is a compound microscope fitted with polarizing prisms, called "Nicols," below and above a rotating circular stage. When two Nicols are placed in the optical train, the first acts as a polarizer and the second as an analyzer. The vibration direction of the plane-polarized light produced by the polarizer is conventionally the north-south direction. If the analyzer, which can be rotated in most instruments, is set in the same relative position as the polarizer (parallel Nicols), then through light is transmitted, producing a light field of view. But, if the analyzer is rotated through 90 degrees so that its plane of vibration is at right angles to the polarizer (crossed Nicols), no light will pass, except that refracted into the analyzer’s plane, producing a dark field of view.
       
      Optically active substances show interference colors when placed between crossed Nicols. Observations of optical activity can be useful to the analyst for identifying to some extent such diverse things as glass fragments, synthetic fibers, crystals, starches, and mites. This segment of training gives the trainee the basic principles of PLM techniques.   Additional descriptions of PLM techniques will be found in Advance techniques Chapter 4.6.2 Optical Crystallography.
   3. Comparison Microscopy (Forensic microscopy)
       
      Comparison Microscopy involves “bridging” the optics of two microscopes, so that they may be viewed independently, side by side, or overlapped for direct comparison purposes. In this manner, filth analysts can compare known specimens, with unknown specimens, and confirm similarities or identify differences.
   4. Lightfield/Darkfield Stereomicroscopes
       
      Lightfield/Darkfield stereomicroscopes are conventional stereomicroscopes mounted on a special light base, the base of which produces either a white background (fully illuminated field of view) or a dark background (with incident light coming in at an angle). The technique is very useful in increasing contrast (similar in a way to phase contrast) and in viewing light subjects against a dark background.  Examples include using darkfield illumination when trying to count mites, versus counting white mites or maggots against a white background.
   5. Scanning Electron Microscopes (SEM)
       
      Scanning electron microscopes have particular application in particle analysis and detailed micro-structural analysis. Several publications describe the use of electron microscopes in the examination of mites and stored product beetle mandibles, antennae and related structures, but due to the high expense of the instrument (initial purchase and upkeep), specialized training, room or space needs and preparation time, it has not been used in most field applications. In the area of particle analysis, augmentation techniques such as x-ray augmentation, help to identify particles not only on their structure and morphological form, but also in their chemical composition. Newer instruments and techniques now allow for what are referred to as “wet” examinations; low vacuum chambers are designed for “wet” mounting live specimens, like mites. Normally, SEM units call for the specimen be mounted on metal studs and then dried to absolute dryness before the specimens are gold sputter coated.
4. Microscope Accessories
   1. Eyepiece Micrometer (or graticule)
      
      The eyepiece micrometer consists of a clear disc with a graduated scale or pattern printed on it, which is inserted in the eyepiece of the microscope for use in measuring specimens. In order to obtain meaningful measurements, the graticule is calibrated for each magnification, by using a stage micrometer with a graduated scale of known increments (usually 0.1 mm for stereomicroscopes or 0.01 mm for compound microscopes). 
      
      Calibration Procedure 
      
      Place the stage micrometer on the microscope stage and focus on the stage micrometer scale. (The eyepiece graticule should always be in focus; if not, the disc is probably improperly inserted or out of adjustment.) Move the stage micrometer so that the zero end of its scale coincides with the zero end of the eyepiece scale and the two scales are superimposed on each other over their entire lengths. Reading from the eyepiece scale, find the farthest division from zero that coincides with a division on the stage micrometer.
      
      Record the following information:
      
      • Magnification.
      • The number of eyepiece scale divisions between the zero coincident and the farthest eyepiece scale coincident. This value is designated EMD (eyepiece micrometer divisions).
      • The number of stage micrometer scale divisions between the zero coincident and the far stage scale coincident. This value is designated SMD (stage micrometer divisions).
      • The millimeter value of one division of the stage near scale. This value is usually found printed on the micrometer. This value is called mm/SMD (millimeters per stage micrometer division).
        
        
      Calculate the millimeters per eyepiece micrometer division (mm/EMD)
      
                   mm/EMD = (mm/SMD) X (SMD)/(EMD) 
       
      The mm/EMD is the number of millimeters per eyepiece micrometer division for that particular magnification. This value is used to convert eyepiece micrometer divisions to millimeters by multiplication. (Note: These calculations need to be determined for each set of eyepiece and objective combinations.)
       
      Example: At 10X magnification, 12 EMD coincide with 18 SMD on a stage micrometer in which 1 SMD = 0.1mm 
       
                   (0.1mm/SMD) X (18 SMD)/(12 EMD) = 0.15 mm/EMD
       
      If an object is observed to be 5 EMD long at 10X magnification, then 
       
                   (0.15 mm/EMD) X (5 EMD) = 0.75 mm
       
      The object's length is calculated as 0.75 mm, the last digit being only an approximation. The significance of calculated numbers should be carefully considered in light of the mathematical rules concerning significant figures.
      
   2. Mechanical Stage Micrometer
       
      This “accessory” consists of two graduated scales that are engraved or inscribed on the mechanical stage. One scale is on the moving portion and the other scale is directly parallel to the first on the stationary portion of the stage. As the length of the specimen's image is moved through a fixed point in the field of view, the number of divisions on the stationary stage scale can be counted by observing the starting and finishing positions of the sliding scale's end point. By substituting this number for the EMD in the equation found in the previous section, the trainee can calculate a conversion factor using a stage micrometer with a graduated scale of known increments. Quite often, mechanical stage micrometers will have a provision for interpolating the final digit of a reading, which adds to their accuracy.
       
      Note: The eyepiece micrometer has more versatility than the mechanical stage micrometer; the eyepiece micrometer can positioned over the image at exactly the angle desired for determining longest and shortest dimensions.
      
   3. Camera Lucida (Abbé type)
       
      The Camera lucida (Abbé type) consists of a set of prisms that can superimpose the image of a specimen onto a piece of paper lying on the bench top beside the microscope. With practice, while viewing the specimen through the scope, the trainee can see the paper and pencil at the same time as the specimen, thus producing accurate outlines or detailed drawings of the specimen under observation. (Hint:  Illuminate the drawing paper with a strong light.) 
       
      This technique is particularly useful when photography does not show the desired details (poor depth of field or cluttered information), that line drawings can produce.

C. Assignment

1. Read the following:
   • Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, decomposition and foreign matter ( FDA Technical Bulletin No. 1, 2nd ed. pp. 219-228). Gaithersburg, MD: AOAC International.
   • Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in the food industry (out of print, FDA Technical Bulletin No. 2, chap. 2).
   • Möllring, F. K. Microscopy from the very beginning (Brochure G41-100, p. 58).  U.S. and Germany: Zeiss, Inc.  Or,  Möllring, F. K. (1973). Beginning with the microscope. New York: Sterling Publishing.  Both out of print, but useful if one can obtain a copy.
   • The microscope manufacturer’s manuals for the scopes the analyst will be using. 
2. Demonstrate the optimal set-up and illumination with specimens provided by the trainer using the following:
   • Widefield stereoscope
   • Compound microscope
   • Phase-contrast microscope
3. Prepare a table of measurements for an eyepiece micrometer on a compound or stereomicroscope in  the laboratory.

4.2.3 Preparing Microscopic Slide Mounts

A. Objective

The trainer provides basic instructions for properly preparing permanent and semi-permanent microscopic slide mounts of hairs, insect fragments, minute whole insects, or other "filth" elements.  Slide mounts may be prepared for use in reference collections or as teaching aids or, in regards to regulatory samples, as evidence in a court of law.
 
This training is applicable to all analysts involved in the identification of filth elements found in foods.  General entomological knowledge and/or training are not a prerequisite for this section.

B. Discussion

1. Introduction
   
   No amount of microscope alignment, focusing, or other manipulation can undo the damage done by the improper mounting of a specimen on a microscope slide.  The mounted filth specimen prepared for regulatory work is an item of evidence, ultimately subject to the scrutiny of a court of law. Fine detail observations may provide significant clues and to the identity of the object. Additionally, the need for quality mounted materials for use in a reference collection is essential, and is tantamount to good museum practice. As such, the analyst strives to become proficient at producing professional-quality slide mounts.
2. Equipment and Reagents
   1. dissection microscope
   2. dissection needles
   3. fine (needle) point forceps
   4. microscope slides
   5. microscope slide cover slips (square and/or round, 1-1½ thickness)
   6. adhesive labels
   7. hot plate
   8. alcohol lamp (IF the laboratory allows open flames)
   9. slide warming plate (typically at 45-50ºC)
   10. water bath
   11. glycerin
   12. gelatin
   13. phenol
   14. gum arabic (crystalline form)
   15. chloral hydrate
   16. distilled water
   17. 2% solution of aqueous acid fuchsin or lignin pink
   18. commercially purchased "permanent" microscope mounting media (i.e. Permount, Euparal, and/or Canada Balsam)
   19. ringing compounds- nail polish (clear preferred) or Glyptal® (electrical insulating varnish)
   20. fume hood
   21. standard safety equipment (laboratory coat, eye protection, gloves)
3. Specimens for Observation
   1. General information
      
      There are many text books and articles written on mounting specimens for observation under the scope. Many techniques date back to the early development of the microscope, as people worked with various formulas to accomplish the perfect slide mount for whatever material they were studying. Over time, people and disciplines developed preferences based on their needs, ease of use, or understanding of the media. Selecting the media of choice varies significantly based on the following factors:
      • How easy is it to use?
      • What is the refractive index and how well does it work with certain specimens of higher or lower index? 
      • What preparation steps are needed?
      • How long does it take to prepare the finished slide? 
      • How long will the slides last - temporary, intermediate, or permanent, before they discolor, crack, or cloud over?
      • What effect does the media have on the specimen or stain specimens?
      • Does it need ringing?
      • Is it expensive?
      • Does it call for the use of noxious chemicals?
      • How valuable is the specimen - what type, an authentic, forensic value as evidence, or quick, non-permanent observation?
        
      Based on these questions this next section offers some discussion of the media FDA analysts have found to be of the most value and the easiest to use.
   2. Media
      
      Glycerin Jelly (GJ) Media
       
      The most commonly used and preferred medium for mounting hairs and insect fragments is glycerin jelly (GJ). 
       
      The formulation is 10 g gelatin, 70 ml glycerin, 60 ml H2O and 1 g phenol.  The gelatin is poured on cold water to soak, and then heated over a water bath to completely dissolve the gelatin.  The glycerin and phenol are mixed while hot.  When cooled, the mixture has the consistency of semi-hardened gelatin and it melts around 35-40ºC. Glycerin jelly can also be ordered from some chemical supply houses.
       
      To mount a specimen, a small piece of glycerin jelly is placed on a slide and warmed to the point where it becomes fluid. As an alternative, the analyst can use pre-melted material, from a glass rod dropping bottle, held on a slide warming plate. With practice, the analyst will have more control of the media droplet size using pre-melted media. The specimen is placed, then pushed into the media with a needle probe into the bottom/center of the liquid medium, then oriented to the desired position, and covered with a coverslip. Warming the slide again is sometimes needed for the jelly to engulf the specimen and fill the space under the coverslip. If trapped air is present near the specimen, the air can be removed by gently heating it over an alcohol lamp or low temperature hot plate.
       
      Caution: This may cause the specimen to migrate to the edge of the coverslip. If this happens, the analyst may need to remount the specimens and make another preparation. It is important for the analyst to practice mounting specimens in order to get a "feel" for the peculiarities of glycerin jelly.
       
      To mount hairs in glycerin jelly, the analyst uses a little extra heat to drive out the air in the center (medullary) portion of the hair.  The characteristics of the hair cannot be observed until the air inside the hair has been replaced with glycerin jelly.  One common method of removing air is to heat the mounted specimen carefully over an alcohol flame or hot plate until the glycerin jelly under the coverslip begins driving the air out of the hair (Note:  just below the media’s boiling point), then cool and observe the specimen at high magnification to see if the air is gone.  Continue heating and observing until sufficient air is driven out to make definite identification.  Again, practice is needed, as too much heat will curl and distort the hair, warp the coverslip, and denature or discolor the medium.  See also Reference:  LIB 2243, “Improved Procedure for Liquid Replacement of Entrapped Air in Mammalian Hairs.” 
       
      Note:  Some analysts make a slide by piling the specimen and coverslip on top of a solid piece of glycerin jelly and then warming the slide so that the glycerin jelly engulfs the specimen.  Two problems may occur using this method.  One, the specimen migrates with the melting media towards the edge; secondly, large air bubbles can be formed and trapped under the coverslip.  These are considered permanent mounts when ringed (ringing is discussed below, Section C). They should be held flat, even when rung. They are stable for at least 5 years and longer if rung, and generally do not discolor or cloud over time. Specimens do not need special drying (water removal) processes as in other resin or Canada balsam mounts, however, mounting from dishes wet with glycerin-alcohol (50/50) or 70% alcohol does reduce some of the trapped air problems. 
       
      Hoyer's Solution
       
      Hoyer's solution is a mounting medium that has been used by entomologists for decades and is now gaining popularity in some areas of food analysis.  It is found commercially, but has many formulation variations, all principally gum-chloral hydrate derivatives. In addition to its excellent optical properties (~1.47), Hoyer’s solution renders muscular and visceral tissues transparent (clearing effect), allowing the analyst to observe cuticular structures on the intact specimen without interference.  This medium is used primarily for mites and small insects, but it can also be used for insect fragments. It is not used for hairs, except as a temporary mount, as it will disintegrate a hair over time. When rung the slides will last several years, but eventually moisture will enter and the slides will cloud over. 
       
      Hoyer's solution consists of 50 ml distilled H2O, 30 g gum arabic, 200 g chloral hydrate, and 20 g glycerin.  The gum arabic should be in crystalline form since the powdered form is difficult to wet.  Ingredients are mixed in the given sequence; allowing time for one ingredient to be completely dissolved before adding the next.  The final product is filtered through bolting cloth or glass wool.  This medium has numerous modifications with names such as Berlese's fluid and de Faure's Fluid.  (Safety note: Care is to be taken with chloral hydrate compounds; breathing the fumes and exposure to the chemical are not recommended. Use only in a hood.)
       
      Specimens may be mounted in Hoyer's solution directly from aqueous solutions or may be mounted live.  This medium has good optical properties for phase-contrast microscopy.  To obtain a longer-lasting slide, the slide mount is cured for 48 hours to one week at 45oC (113°F) and then held at room temperature for one week before sealing.  Slides left undisturbed at relatively uniform room temperature will cure naturally in about three to four weeks.  Temperatures above 45oC will harm the medium.  Whenever using this medium, care is taken to properly vent fumes.
       
      Canada Balsam, Permount, Euparal
       
      For permanent slide mounts, Canada balsam is the most commonly recognized medium. Other commercially found mountants include Permount® and Euparal. They are desirable for museum quality work and for extremely long term storage of authentic materials. All of these are natural or synthetic resin based mountants. The major drawback to these materials is timeliness and almost all typically call for tedious and often difficult specialized water removal drying techniques to prevent clouding. The drying techniques employ a series of gradient alcohols, to xylene, to mixed xylene/mountant solutions, prior to mounting in the diluted resin. Euparal, an alcohol based mountant, is an exception to the full xylene based systems, but it still calls for gradient alcohol fixing stages.  For purposes of this training, these mountants demand advanced techniques beyond the scope of this section, however students should be aware of their usefulness and need for long term storage. Note: Use xylene and toluene in vented areas only. Histological hoods are recommended if these compounds are used with any frequency.
   3. Ringing (Preserving Slides)
      
      Preservation of slide mounts is accomplished through a technique referred to as ringing.  Glycerin jelly and Hoyer's solution are primarily media for nonpermanent slide mounts.  However, with careful preparation and maintenance, they can be made to last many years.  Once the medium has set (hardened), the coverslip edges should be sealed to prevent moisture exchange and to hold the coverslip in place.  The most common sealants are nail polish or Glyptal, (a flexible sealant for electrical connections). The sealant is painted on with a small brush. Round cover slips slides are typically centrally mounted on a rotating table (like a Petri dish turntable). The table is spun, and while holding the brush steady overhead, the sealant is applied around the edge as the coverslip rotates underneath.
   4. Clearing (Removal of Interfering Material)
       
      Clearing is a process that clears, removes or dissolves excess proteinaceous, gut or optically interfering materials from specimens.  Clearing renders a specimen more optically usable for mounting, as a cleared specimen often shows more detail and surface characteristics. The most common method places the specimen in a sodium or potassium hydroxide solution (5-10%), and gently heating the solution until one produces the desired clearing effect.  Lactic acid and lactophenol are also useful for soft-bodied specimens. 
       
      Since this is a destructive process, do not let the specimen stay too long in the solution. Consideration should be given to neutralizing the hydrolyzed specimens, to prevent undesirable continued hydrolysis compatibility problems with subsequent mounting media.
       
      This technique is not to be confused with bleaching, which removes excess pigmentation and coloration.  However, some bleaching action may still occur in cleared specimens.
   5. Staining Specimens
       
      After clearing, most small arthropods are more clearly observed with differential staining.  Two percent solutions of aqueous acid fuchsin or aqueous lignin pink have been used successfully on aphids, mites and Collembola.
   6. Specimen Orientation on Slides
       
      Specimens being mounted for examination under a compound microscope should be mounted in the middle, oriented with the head or front end directed towards the bottom of the slide, and centered under the coverslip. Normally, mount one specimen per slide. However, there will be situations when it is acceptable to mount multiple specimens on one slide.  Examples include similar items mounted next to each other to show similarities or differences, sex differentiation in the same species, or to demonstrate different orientations of the same species. When mounting multiple specimens, keep in mind labeling space and the added difficulties of orienting multiple objects without them moving from the desirable positions.
   7. Labeling Slides
       
      Promptly identify every slide. Label the right side (lot label) with the sample number, sub number, date, analyst's name or initials. If space allows include the product, country of origin, lot or location. Label the left side with the specimen identification (to the correct taxonomy level) and include any additional information that is useful, e.g. sex, stage, size, and fragment. The identifier’s name or initials and date, if different from the preparer, should be on this label. Either side may include the mounting medium or ringing material, to facilitate later remounting.
   8. Storage of Slides
       
      Slide mounts should be stored flat. The mounting media may retain a small amount of fluidity if excessive media is used, or if it is not properly cured or rung. Over time, gravity may cause the coverslip or the specimen to migrate downward if held vertically. Protect the slide from crushing or accidental inversion. 

C. Exercise

1. Prepare the following:
   1. Glycerin jelly
   2. Hoyer's solution
2. Properly mount and label specimens of:
   1. Any whole insect which measures less than 3mm
   2. Insect fragment(s) elytra, a pair of mandibles, legs
   3. A mouse hair
   4. A mite

D. References and Supporting Documents

• Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in the food industry (out of print, FDA Technical Bulletin No. 2).
• Martin, J. E. H. (1977). Collecting, Preparing and Preserving Insects, Mites and Spiders (The Insects and Arachnids of Canada Series, Part 1).  Ottawa: Agriculture Canada.
• Uribe, M., Vail, D. J. (1980). Improved procedure for liquid replacement of entrapped air in mammalian hairs. FDA Laboratory information bulletin,  No. 2243.
• Bullington, S. W.  How to mount maggots on microscope slides. Forensic Entomology website: http://www.forensic-ent.com/.
• Schauff, M. E. Collecting and preserving insects and mites: techniques and tools. Washington, D.C.: Systematic Entomology Laboratory, USDA, National Museum of Natural History. http://www.sel.barc.usda.gov/selhome/collpres/collpres.pdf.
• Horobin, R. W., Kiernan, J. A. (Eds.). (2002). Conn's biological stains: a handbook of dyes, stains and fluorochromes for use in biology and medicine (1st ed.). Abingdon, UK: Bios Scientific Publishing, Ltd.

4.2.4 Collecting and Preserving Whole Insects and Arthropods

A. Objective

This section will provide basic instruction for collection and preservation techniques for whole insect and arthropod specimens found in regulatory samples and those specimens collected for the laboratory's authentic reference collection. A filth analyst does not routinely collect insects in the traditional sense (for example with a butterfly-net), and we rarely preserve them as a pinned specimen. Although the majority of the laboratory’s work will deal with insect fragments, whole specimens are first discussed. Analysts do find whole insects, (and in some cases, whole live insects) in regulatory samples.  These insects are evidence that are properly preserved for courtroom presentation. If the specimens are collected for use as authentics, as reference materials and/or direct comparison with unknown specimens, only the best museum preservation techniques are applied. 

B. Procedure

1. Recommended Equipment
   1. Vials (1/2 dram, 1 dram, and/or 2 dram sizes, glass with screw-on cap with polyseal® cone insert recommended)
   2. Sieves (U.S. Standard #8, #20, #40 and pan; a "collar" is also recommended)
   3. Berlese Funnel and/or Tullgren Funnel
   4. Aspirator (recommend "exhalation" style or "inhalation" style with in-line filter)
   5. Artist's camel hair brush (recommend #2 or #3)
   6. Jeweler's or needle point forceps
   7. Flexible steel forceps
   8. 5X Magnifying lamp
   9. White plasticized butcher paper
   10. Various sized white or stainless steel pans
2. Recommended Reagents
   1. 95% Ethanol
   2. Glacial acetic acid
   3. Kerosene
   4. Dioxane ("Triton X-100" or "Tween 80" may be substituted)
   5. Formalin (40% formaldehyde)
   6. 10% KOH, 10% NaOH, 50-85% lactic acid, or 2:1:1 lacto-phenol
   7. Distilled Water
   8. Ethyl acetate
   
   Note: There are commercial killing fluids, clearing agents, and preservatives.  If these commercial products are used, the above list of reagents could be reduced.
3. Collecting Insects
   
   Insects found in regulatory samples by macroscopic examination or microscopic examination are collected and preserved as evidence.  In some cases, these insects may be alive.  The method used to extract insects from a product will vary based on the composition of the product.  Although a method may be dictated, there are some "traditional" entomological methods that may be of value.
   1. Sieves
       
      Insects may be removed from some products by placing the product in nested sieves (generally, a #8 is placed over a #20 or finer.)  If the product being sieved needs to be contained, a collar or lid is added to the uppermost sieve, and a pan to the lower.  Generally, the product is placed in the uppermost sieve and shaken, causing insects and other foreign material to fall onto the lower sieve and into the pan.  Conversely, if the filth analysis is looking for larva in flour, the larva is retained in the uppermost sieve, and the flour passes through to the pan below.
   2. Berlese/Tullgren Funnel
       
      The Berlese Funnel, originally developed by Antonio Berlese to remove live mites from leaf litter, is an option for removing live insects and mites from reasonably dry leafy food materials (i.e. taro or palm leaf).  The device is a large funnel with a coarse (U.S. STD #2, #4, or #8) woven metal screen inserted above the neck to hold back the product, yet allow insects to pass through into a jar of preserving fluid.  Insects and mites will be driven down by a heat source (generally a 40-60 watt light bulb) in a lid that covers the mouth of the funnel.  The Tullgren Funnel, a modification using a series of baffles rather than a screen, has been used to remove insects and mites from dry powders or granules that are too small for a typical Berlese.
   3. Aspiration
       
      An aspirator may be used to vacuum up small insects and/or mites.  One type of aspirator involves inhalation or a vacuum pump to draw the insects into the container.  Another type uses exhalation or an air pump to collect the specimen.  It is recommended if an inhalation type aspirator without a vacuum pump is used, insure that there is an in-line filter between the user and the collection chamber, in particular where the substrate is harmful (from microbes, spores, or chemicals.)
   4. Killing Insects
      1. Freezing
         
         When dealing with live insects or where insects are in dry products, freezing is the easiest and safest technique, provided the analyst allows time for proper penetration of the cold temperatures to the center of the product or exhibit. This can be checked with a thermometer. During the freezing process, insects are driven away from the cold temperatures and into the center of the product or exhibit. Care is taken to maintain the specimens in a frozen condition, until they can be examined. This is needed because allowing them to come back to room temperature for extended periods of time (e.g. a day), will cause discoloration and damage to a specimen.  Freezing does not stop enzymatic and gut microbes from continuing their actions inside the warmed specimen. Once frozen, the specimens can be manipulated and picked out of the exhibits for preservation as identified below.
      2. Killing Fluids
         
         Immersing a larva in hot (near boiling) water is the best killing fluid for larvae in a laboratory situation.  This treatment stops enzymatic and gut microbe action and distends the larva. The larva is then removed and put into cold water, or directly into 70% alcohol. Killing fluids such as KAAD (a mixture of kerosene (1 part), ethanol (7-10 parts), glacial acetic acid (2 parts), and dioxane (1 part)) may be used to kill and "fix" a larva to avoid discoloration or distortion.  The killed larvae will need to be removed from the KAAD solution and transferred to a preservative within 24 hours.  
          
         Safety Note: Dioxane may become unstable if stored more then 12 months.  Use of "Triton X-100" or "Tween" is recommended instead.
      3. Fumigants
         
         Generally used for adult insects, a fumigant is a substance that generates a poisonous gas.  
          
         Fumigants of choice in a filth laboratory are quick, easy to use and lethal to insects and mites, but relatively safe for the analyst. 
          
         Liquid fumigants are more common, (e.g. ethyl acetate); a small amount of the liquid can be placed on an absorbent pad and placed in an airtight container or killing jar.  Note:  Cotton balls are NOT recommended as the absorbent pad, as insects may become entangled in the fibers.
          
         Solid form fumigants (e.g. paradichlorobenzene) may be used; the solid form is held inside a screened chamber built into the lid of the killing jar.  (Note:  Paradichloro-benzene is a slow acting fumigant.) 
          
         In both cases, the insect is placed into the airtight container with fumigant and asphyxiated.
         
         Safety Note:  Numerous fumigants are used, but all generally have higher human health risks. Regardless of the fumigant used, the analyst works in a well-ventilated area or fume hood; care should be used to avoid breathing the fumes.  
          
         Caution:  Ethyl acetate and other fumigants may have solvent actions that may dissolve the container, lid, or seals. Always check compatibility first, before using solvents.
   5. Preserving Insects
      1. Preservatives
         
         Most specimens are stored in poly-cone capped glass vials of 70% ethanol, others (soft bodied maggots) in a 1:1 of 70% ethanol and glycerin, some (insect eggs) in pure glycerin, and still others (pigmented soft bodied) in commercially prepared solutions such as Pampel's or Kahle's.  Mites are generally stored in 70% ethanol, lactic acid, or in AGA (87 parts 70% ethanol, 8 parts glacial acetic acid and 5 parts glycerin). 
          
         The analyst is reminded that some preservation agents, like formaldehyde, are also fixatives. They may actually cause damage to the specimens through subtle color loss or by tissue shrinkage from the denaturing of the proteins. Analysts are encouraged to ask the senior analyst questions as the need arises, or consult the references cited below or in the Reference Appendix.
      2. Pinning and Air Drying
         
         Pinning, spreading, and air drying are techniques commonly used for whole adult insects that are generally placed in museum boxes. Smaller insects (too small to be properly pinned) can be placed (glued) onto triangular shaped paper points, the points of which are then mounted on pins. This technique is commonly used for authentic specimens, where handling is minimized and easy access to dry specimens is needed.  Normally, given the size of most stored product insects and their brittle nature when pinned, we do not preserve regulatory sample specimens by pinning them, as the specimens are too fragile to stand much handling, especially where handling is out of the analyst’s control. However, some flies and thin cuticle specimens are better preserved on pins and careful packaging measures would be needed to assure that the pinned regulatory specimens are not damaged from dropping, crushing, or shaking of the finished exhibits.
         
         See Schauff’s document at http://www.sel.barc.usda.gov/selhome/collpres/collpres.pdf for details on pinning specimens for the best results.
   6. Labeling Preserved Specimens
      
      Every specimen (or group of like specimens) collected are to be labeled immediately. The value of a specimen is seriously diminished if the specimen is left unlabeled or incompletely labeled. 
       
      Depending upon the type of preservation method used, labels should be on acid-free 28-60# index-weight paper using indelible ink or laser printing. Inkjet printing is not usable for wet vialed specimens. The labels can consist of one or more (broken up into sample collector’s or lot label(s) and identification label as grouped below) labels and they are placed in the vial or dish or on the pin with the specimen(s). The complete label should include the following information: 
       
      Group 1
      1. Sample number (Lot)
      2. Sub number
      3. Date collected (or date extracted by analyst)
      4. Collector's  or Analyst's name or initials
      5. Type of preservation fluid (if applicable)
      
      Group 2
      6. Product
      7. Country of Origin
         
         
      Group 3 
      8. Identification of specimen
      9. Who identified specimen and date

C. Supporting Documents

1. AOAC official methods of analysis (current Ed.). Gaithersburg, MD: AOAC International.
2. U.S. Food & Drug Administration, Center for Food Safety and Applied Nutrition. (1998). Macroanalytical procedures manual (FDA Technical Bulletin No. 5). Retrieve 1998 electronic version from http://vm.cfsan.fda.gov/~dms/mpm-toc.html  [Originally released in print by FDA in 1984 by Olsen, A. R. (Ed.), Knight, S. A. (Tech. Ed.), Ziobro, G. C., Ph.D. (Assoc. Ed.)] 
3. Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in the food industry (out of print, FDA Technical Bulletin No. 2, HHS Publication No. (FDA) 77-2086). 
4. Stehr, F. S. (1987). Immature insects (Vol. I). Dubuque, IA: Kendall/Hunt Publishing Co.
5. Schauff, M. E. Collecting and preserving insects and mites: techniques and tools. Washington, D.C.: Systematic Entomology Laboratory, USDA, National Museum of Natural History. http://www.sel.barc.usda.gov/selhome/collpres/collpres.pdf .
6. Vazquez, A. W. Examination of bulk samples of food products for infestations with living insect and mites using the Berlese funnel technique. FDA Laboratory Information Bulletin, No. 883. 
7. DeCamp, P. W. Modifications of the Berlese funnel for filth work. FDA Laboratory Information Bulletin,No.1816. 
8. Biological Survey of Canada. (2001). Brief: Label data standards for terrestrial arthropods. http://www.biology.ualberta.ca/bsc/briefs/brlabelstandards.htm.

D. Glossary

Collar - An old sieve with the weave removed; extends the height of a sieve.

Fix - Chemical process that prevents or minimizes pigment discoloration, loss and/or tissue distortion, to preserve in place.

Overs/Throughs - After a sieving operation, anything retained on top of the screen is referred to as overs, those that pass through, as throughs.

E. Exercises

1. Prepare the following solutions:
   1. 60% ethanol
   2. Glycerin-Alcohol (1:1)
   3. A.G.A.
   4. 10% KOH
   5. KAAD
2. Kill and preserve the following, including a label:
   1. adult stored product beetle (for regulatory sample, for authentic use)
   2. larval stored product insect (for regulatory sample)
   3. a mite 
   4. a cockroach
   5. a spider

4.2.5 Taxonomy

4.2.5.1 Taxonomy Principles

A. Objective

This exercise will provide background in taxonomic principles and zoological nomenclature.

B. Discussion

Taxonomy is the science of giving names to organisms in order to classify them. The system used to name organisms was designed by the 18th century botanist, Linnaeus. It consists of a basic name for each kind of organism and a hierarchy of categories for grouping similar kinds of organisms together. Identifying an organism, then, is simply finding the proper name to call it.

The basic name or scientific name of an insect or any other biological entity consists of two Latin or Greek based words. Each name combination is unique; there is no duplication of names under the system of Linnaeus. Scientific names also have a strict format. The format is as follows: 

The basic unit of the system is the species. Next is genus, a group of species that is closely related phylogenetically (by ancestry). The system continues building larger and larger categories, each indicating a more remote phylogenetic relationship. The general progression of categories is given below with examples of each category.

Apis mellifera (Linnaeus) is the scientific name of the insect commonly called the "honeybee." In order to be sure however, that this is the correct name for the insect in question, a test is applied. For insects, and most other organisms, the test compares a specimen to either a validated (or authenticated) specimen whose identity is assured, or to compare the specimen to a written description of the validated specimen. In FDA work, both methods are used to confirm the identity of an insect.

In entomology, "validated" specimens are called "type" specimens. Type specimens are specimens that have been designated as examples of a particular species by the scientist who originally named the species (the author). In FDA, "authentic" specimens are similar to type specimens in that they have been verified by experts to be good examples of a particular species of insect.
 
Technical descriptions are written, detailed descriptions of a type specimen. Since most type specimens are housed in large museum collections and not provided for casual examination, published technical descriptions are needed and useful. Although we may not have the original author’s description, most district laboratories will have literature containing technical descriptions of common food-infesting insects, which we use to make identifications.

C. Assignment

1. Examine the laboratory collection of authentic whole insect specimens.
   
2. Determine the books or journals that contain technical descriptions of stored-product (food-infesting) insects.

D. Questions

1. Give a literature reference for a technical description of the coffee bean weevil, Araecerus fasiculatus (DeGeer).
   
2. What is the full scientific name, including author, of the confused flour beetle? 
   
3. Based on the scientific names of the insects in "a" and "b," would one expect them to be similar to each other or dissimilar?

4.2.5.2 Identification Keys

A. Objective

The trainee will learn how to use identification keys.

B. Discussion

A key is a guide to the identification of an insect. Although there are various kinds of keys, each kind attempt to arrange the characteristics of a particular group of insects into an orderly format with the intent of guiding the analyst through a series of observations until every species but one is eliminated. Keys are not infallible, final, or all-inclusive.   Keys provide a tentative answer; the final identification of an insect depends on direct comparisons with technical descriptions and authentic specimens. The value of a key tells the entomologist which technical descriptions and authentic specimens to look at first, and helps narrow down the search in a structured fashion.

Dichotomous keys are the most common entomological keys. Their basic composition is a series of pairs of mutually exclusive statements about the specimen being identified, called couplets. By choosing the statement that best satisfies the insect being observed, the entomologist is directed to another couplet. This process continues until a couplet is reached that indicates a name (typically to the species level).  That species name is the most likely identification of the insect.  However, FDA demands the analyst confirm this name identification with authenticated material, or at least to a literature description.

There are variations on the basic dichotomous key format. Sometimes a key will include a triplet (three mutually exclusive statements) or even a quadruplet. In these cases, the entomologist still chooses only one statement. As a kindness to identifiers who occasionally backtrack, lengthy keys will often provide parenthetical reference to the previous couplet immediately following each couplet number.

Although dichotomous keys are often illustrated, pictorial keys rely on illustrations to guide the entomologist towards an identification. Using a series of illustrations with terse legends, the pictorial key guides the entomologist by directional arrows, much like an agency personnel table of organization.

Tabular keys are useful for distinguishing members of a small group of similar appearing insects. A table compares the distinguishing characteristics of each insect. Frequently, a single characteristic may be duplicated or characteristics may be overlapping, but each species will have a unique total set of characteristics that will distinguish it from others.

Hint: Experience shows that the analyst doesn’t always get the answer expected when using keys, especially if the keys are complicated, or some of the characteristics are ambiguous. To help remedy mistakes, and keep from going back to the beginning, on a separate piece of paper, try keeping a running list of choices, i.e. 1, 3, 4, 17, 18, 23, 30 etc., and circle the couplets that are major breaking or grouping points in the key. Also, put a question mark (?) above the ambiguous or questionable couplet choices. This will allow one to retrace steps and review decisions. This will save a lot of time, especially in unfamiliar territory.

C. Assignment

1. Examine one or more examples of each of the three types of keys: dichotomous, pictorial, and tabular. 
   
2. The trainer presents a set of objects.  Construct a simple dichotomous key.  
   
3. The trainer provides an unknown specimen(s).  (Beginner Level).  Identify the specimen(s), using each of the three types of keys.

4.2.6 Digital Photography and Photomicrography

A. Objective

This procedure applies to all analysts using digital cameras or scanners for photodocumentation of evidence/sample casework. This procedure is written for film-less photography with images stored on magnetic or optical (CD) media.

B. Definitions and Acronyms

Aperture - Circular hole in the camera that controls the amount of light reaching the sensor or film emulsion.

Blooming - The bleeding of signal charge from extremely bright pixels resulting in over-saturated pixels. “Blooming” in digital photography compares with over-exposure in film photography.

BMP - Bitmap (.BMP file extension): this is a standard image file format for Windows®.

GIF - Graphic Image Format (.GIF file extension): gif format is what is termed as a "lossless" compression format.  This format is referred to as a "paletted" image or a 256 color image. It is limited to 256 colors.

JPEG - Joint Photographic Experts Group or jpeg (.JPG file extension) this format is a lossy compression format. The higher the compression ratio the more the pixelization or "blockiness" occurs.

Cropping - The act of cutting out a portion of a digital image for blow-up/display as a separate image. 

Photodocumentation - The process of recording images representative or demonstrative of a sample or object.

PNG - Portable Network Graphics (.PNG file extension): this file format is an alternative to the GIF (Graphic Image Format) format.

TIFF - Tagged-Image File Format (.TIF file extension): a lossless bitmap image format supported by virtually all paint, image-editing, and page-layout applications.

C. Discussion

The replacement of film emulsions with digital sensors for imaging brings many conveniences to laboratory photodocumentation.  Digital imaging provides an instant review of composition for quality and facilitates archival through modern digital storage techniques.

Due to variations in equipment from laboratory to laboratory, the trainee learns to use equipment with the guidance of an analyst familiar with its use. As with conventional film photography, it is important to pay attention to the composition, lighting, and overall contrast of a scene when photographing samples.  A well-composed picture has the following qualities:

• The subject is positioned and contrasted to attract the viewer's attention. 
• Important details are highlighted. 
• The subject is magnified so it is not lost as an inconspicuous speck in the photographic field. 
• Sample identification is included in the scene, if possible
• When possible, a scale of known distance intervals is included in all images. If not possible, all photographic conditions, including magnification and lenses used, are recorded and a calibrated bar marker inserted in the photomicrograph.

For digital photographs or scans, the following equipment (if used) should be documented on the worksheet or a separate attachment:

• Brand and Model of the Digital/Video camera 
• Image Capturing device
• Scanner type 
• Printer Brand and Model
• Microscope(s) 
• Illuminator(s) 
• Lens Brand and Model
• Focal Length and Aperture Site
• Exposure time (if applicable)
• Filters (if applicable)
• Image storage (location, file names, etc.)

Digital cameras often have numerous settings that can be set to automatic or manual modes of operation.  Such features will usually call for an SOP, tailored to the equipment, that outlines which settings should be left as automatic and which should be set manually so as to maximize quality, repeatability, and to facilitate in archival.

Original images are not be modified.  When an image is modified (contrast/color adjustments, sharpening, or cropping for instance) a copy of the original file as obtained from the camera is kept.  The new, modified file should be named by appending the word “modified” to the original file name. It is vitally important to document what adjustments were made to the original file in creating the modified one.  A print of the original image alongside or below the modified image may not be needed if the analyst has safely stored the original file.

Both the modified and unmodified files should be written to media for submission with the analyst report.  Each print of an image should be identified with the image name, description, and variables recorded at the time of capture, as previously described.

1. Considerations
   1. Printer
      
      Only paper recognized by the printer manufacturer as fade resistant and fade resistant ink (cartridges) are used. Printer paper stack should be covered when not in use. Printer heads cleaning is conducted as needed. (See instructions with printer manual). 
      
   2. Camera
       
      Mount the camera on a tripod and set the exposure manually by adjusting the aperture so as to maximize depth of field.  Set the image quality to maximum and turn off (or manually set) as many automatic features as possible so as to increase repeatability/consistency.  In particular, in-camera sharpening, white balance, and use of a flash (as well as flash intensity) should be fixed.  Choose a lossless file format if possible; an option to produce a TIFF file is often provided and is desirable seeing as how the TIFF format uses a lossless compression scheme (or no compression at all).
   3. Scanner 
       
      Output Resolution should be set to a minimum 150DPI*.  Sharpening level should be set to low or none (some scanners automatically sharpen a scan).  Output Dimensions: the file format chosen should use lossless compression, or none, as described in step (a).
       
      * The concept of DPI (dots per inch) is often confused with camera, or scanner, resolution.  In reality the two terms are not interchangeable.  Resolution as it pertains to digital imaging refers to the number of pixels captured by the sensor while DPI is a display (i.e. print) characteristic which indicates how many of those pixels are displayed per inch.  Thus, the DPI of an image may be adjusted to no end without affecting the amount of data (pixels) in an image.  The human eye is incapable of perceiving more than 340 pixels per inch (approximate) at a viewing distance of 10 inches.  In practice, a print with a DPI of 150 pixels (dots) per inch or greater will appear acceptable.  As an example, the analyst is to use the maximum resolution of a Nikon Coolpix 4500 digital camera to determine what the DPI of an uncropped image should be set to for printing on an 8.5x11inch sheet of Photo Quality Inkjet Paper:
       
      The resolution of an uncropped Coolpix 4500 image is 1704 pixels wide x 2272 pixels high (using a portrait aspect ratio).  Due to printer margins, we could assume the maximum printable height of the image on the paper will be 10 inches.  The equation describing the relationship between resolution and DPI in the vertical dimension is thus 2272/n = 10.  Solving for n, we see that approximately 228 DPI is needed.  If we carry this same DPI over to the width, we see that the image will be 1704/228 = 7.47 inches wide.  Thus, the solution is to adjust the DPI of the image to 228 before printing.  This is greater than 150DPI so, for most subjects, this print should appear acceptable.  This 150DPI rule of thumb is common in the graphics profession.
       
      Adjusting the DPI as in this example does not change the amount of data in the image since no pixels are being added or removed.  By default, some image editing applications will inappropriately “resample” the image (i.e. interpolate to increase or reduce the number of pixels) when given the command to change the DPI.  Ensure that this is not occurring by checking the number of pixels in both dimensions before and after the DPI adjustment.
2. Acquiring  and Saving Images
   
   1. A scale (ruler) should be positioned in the field of view when a camera or scanner is used to capture an image.  A proportional scale may then be used in the worksheet to provide distance and size information.
   2. Avoid “blooming” an image by checking the exposure prior to capture.  For many scanners this entails running a test scan so as to allow an automatic calibration.  The reflectance properties of some surfaces may need manual exposure adjustments.  For digital cameras, ideal exposures are often a result of experience or trial-and-error.  The LCD preview screen on the rear of most digital cameras can provide some indication of the effectiveness of an exposure, but such displays are un-calibrated and are highly dependent on ambient lighting or brightness/contrast controls.  As a result, apart from providing a check for sharpness they may be far less useful than at first imagined.  One exception is the display of tonal or color information in the form of histograms by some cameras.  Such histograms can provide detailed exposure information when interpreted correctly.
   3. Images are saved on removable media.  Image storage on the local hard disk should only be temporary; there is no need to retain copies of images on a local computer or server once they have been written to the media to be submitted with the worksheet (multiple analysts doing so would quickly overwhelm the storage capacity of the server or perhaps even a central computer designated for managing digital photography).  A preferred medium for storing images is the CD-R disk.  Such disks can be “closed” after being written to and thus offer an unalterable means of storage with the added benefit of tolerance to environmental conditions that would otherwise destroy data on common magnetically-based media.
3. Printing 

Images are to be printed on photographic quality paper using the printer’s highest quality and resolution.  Often the best paper for a given situation will be branded by the printer manufacturer itself.  Specialty papers should be covered when not in use.  Printer head cleaning is conducted as needed with periodic checks to counter nozzle clogging.

A photograph does not replace written results and descriptions. Normally FDA does not require the photographic documentation of negative results.  In short, use discretion.  A photograph can be particularly useful, for example, when it can demonstrate the lack of an item that should be present in a product.

D. Assignment

Read a suitable introductory text; Low, A. (1991).  Introductory computer vision and image processing. New York: McGraw-Hill Book Co. Ltd. 

4.2.7 Analytical Filth Worksheet

A. Objective

To acquaint the trainee with the analytical worksheet (form FD-431) and other standard form worksheets, emphasizing proper presentation of analytical results.

B. Assignment

1. Read the AOAC Official Methods of Analysis, current edition, Chapter 16 on "Light and Heavy Filth;" and see the reporting format described in Chapter 5 of the Macroanalytical Procedures Manual for various products (in particular the 10 sub dried peas and beans, sequential sampling plan for nut products, and others selected by the trainer).
2. Learn the procedures for completing filth analytical worksheets.
3. Examine recent filth food sample worksheets, and review the laboratory’s various forms and formats used to report filth results.